The overall aim of this project is to develop a disease-relevant HTS assay for the discovery of anti-inflammatory drugs to manage chronic lung inflammation in Cystic Fibrosis (CF). The phenotypic hallmark of CF is chronic lung infection from opportunistic microbes such as Pseudomonas aeruginosa. Resultant inflammation eventually leads to deterioration of lung function and death in afflicted individuals. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most inflammatory agents currently used in patients have severe limitations due to adverse side effects. Several studies indicate that the toll-like receptor pathway (TLR) mediates bacterial infection-induced inflammation in CF through hyper-activation of the transcription factor nuclear factor-kappa B (NF?B). Thus, we will develop and optimize a Pseudomonas aeruginosa bacterial filtrate (PAF)-induced image-based translocation assay for the NF?B subunit p65 in CF airway epithelial cells. We have already created and partially characterized a CF airway cell-line stably expressing the p65 subunit of NF?B fused to green fluorescent protein (GFP-p65). Our preliminary data indicate that GFP-p65 is functional in the engineered cells and translocates to the nucleus upon stimulation with PAF. We will develop a comprehensive strategy for imaging these cells in a format suitable for HTS. The assay will be further validated and optimized for HTS through the screening of a pilot library of pharmacologically active molecules. We will also develop and test a strategy to prioritize active compounds from HTS efforts based upon activity and pathway selectivity. Finally, we will define and test our approach to determine the molecular mechanism of action of a test agent and reveal its intracellular targets.